Particle delivery techniques

ABSTRACT

A method is provided for in vivo or ex vivo delivery of a preparation of powdered nucleic acid molecules into vertebrate tissue for transformation of cells in the tissue using needleless injection techniques. The method can be used to deliver therapeutically relevant nucleotide sequences to cells in mammalian tissue to provide gene therapy, elicit immunity or to provide antisense or ribozyme functions. A method for providing densified processed pharmaceutical compositions is also described. The method is used to convert non-dense pharmaceutical powders or particulate formulations into densified particles optimally suited for transdermal delivery using a needleless syringe. The method is also used to optimize the density and particle size of powders and particulate formulations for subsequent transdermal delivery thereof. Densified pharmaceutical compositions formed by the present methods are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 09/216,641,filed Dec. 17, 1998, now allowed, which is a continuation-in-part ofInternational Patent Application Numbers PCT/GB97/01636, filed Jun. 17,1997, and PCT/GB97/02478, filed Sep. 11, 1997, both designating theUnited States, from which applications priority is claimed pursuant to35 U.S.C. §365(c), and which applications are incorporated herein byreference in their entirety.

TECHNICAL FIELD

The present invention relates generally to particle delivery methods.More particularly, the invention pertains to in vivo and ex vivodelivery of powdered nucleic acid molecules into mammalian tissue usingneedleless injection techniques. The invention also relates to methodsfor forming dense, substantially solid particles from non-denseparticulate pharmaceutical compositions such as those prepared usingfreeze-drying or spray drying techniques. The densified compositionsobtained using the method are particularly suitable for transdermalparticle delivery from a needleless syringe system.

BACKGROUND OF THE INVENTION

The ability to deliver agents into and through skin surfaces(transdermal delivery) provides many advantages over oral or otherparenteral delivery techniques. In particular, transdermal deliveryprovides a safe, convenient and noninvasive alternative to traditionaldrug administration systems, conveniently avoiding the major problemsassociated with oral delivery, e.g., variable rates of absorption andmetabolism, gastrointestinal irritation and/or bitter or unpleasant drugtastes. Transdermal delivery also avoids problems associated withtraditional needle and syringe delivery, e.g., needle pain, the risk ofintroducing infection to treated individuals, the risk of contaminationor infection of health care workers caused by accidental needle-sticksand the disposal of used needles. In addition, such delivery affords ahigh degree of control over blood concentrations of administered drugs.

However, despite its clear advantages, transdermal drug deliverypresents a number of its own inherent logistical problems. The passivedelivery of drugs through intact skin necessarily entails the transportof molecules through a number of structurally different tissues,including the stratum corneum, the viable epidermis, the papillarydermis, and the capillary walls in order for the drug to gain entry intothe blood or lymph system. Transdermal delivery systems must thereforebe able to overcome the various resistances presented by each type oftissue. In light of the above, a number of alternatives to passivetransdermal delivery have been developed. These alternatives include theuse of skin penetration enhancing agents, or “permeation enhancers,” toincrease skin permeability, as well as non-chemical modes such as theuse of iontophoresis, electroporation or ultrasound. However, suchtechniques often give rise to unwanted side effects, such as skinirritation or sensitization. Thus, the number of drugs that can besafely and effectively administered using traditional transdermaldelivery methods has remained limited.

More recently, a novel transdermal delivery system that entails the useof a needleless syringe to fire solid drug-containing particles incontrolled doses into and through intact skin has been described. Inparticular, commonly owned U.S. Pat. No. 5,630,796 to Bellhouse et al.describes a needleless syringe that delivers pharmaceutical particlesentrained in a supersonic gas flow. The needleless syringe is used fortransdermal delivery of powdered drug compounds and compositions, fordelivery of genetic material into living cells (e.g., gene therapy) ornucleic acid immunization, and for the delivery of biopharmaceuticals toskin, muscle, blood or lymph. The needleless syringe can also be used inconjunction with surgery to deliver drugs and biologics to organsurfaces, solid tumors and/or to surgical cavities (e.g., tumor beds orcavities after tumor resection). In theory, practically anypharmaceutical agent that can be prepared in a substantially solid,particulate form can be safely and easily delivered using such devices.

One particular needleless syringe generally comprises an elongatetubular nozzle having a rupturable membrane initially closing thepassage through the nozzle and arranged substantially adjacent to theupstream end of the nozzle. Particles of a therapeutic agent to bedelivered are disposed adjacent to the rupturable membrane and aredelivered using an energizing means which applies a gaseous pressure tothe upstream side of the membrane sufficient to burst the membrane andproduce a supersonic gas flow (entraining the pharmaceutical particles)through the nozzle for delivery from the downstream end thereof. Theparticles can thus be delivered from the needleless syringe at deliveryvelocities as high as Mach 1 to Mach 8, which velocities are readilyobtainable upon the bursting of the rupturable membrane.

Another needleless syringe configuration generally includes the sameelements as described above, except that instead of having thepharmaceutical particles entrained within a gas flow, the downstream endof the nozzle is provided with a diaphragm which is moveable between aresting “inverted” position (in which the diaphragm presents a concavityon the downstream face to contain the pharmaceutical particles) and an“everted” position (in which the diaphragm is outwardly convex on thedownstream face as a result of a supersonic shockwave having beenapplied to the upstream face of the diaphragm). In this manner, thepharmaceutical particles contained within the concavity of the diaphragmare expelled at a high initial velocity from the device for transdermaldelivery thereof to a targeted tissue surface.

Transdermal delivery using the above-described needleless syringeconfigurations is carried out with particles having an approximate sizethat generally ranges between 0.1 and 250 μm. For drug delivery, atypical particle size is usually at least about 10 to 15 μm (the size ofa typical cell). For gene delivery, a typical particle size is generallysubstantially smaller than 10 μm. Particles larger than about 250 μm canalso be delivered from the device, with the upper limitation being thepoint at which the size of the particles would cause untoward damage tothe skin cells. The actual distance which the delivered particles willpenetrate depends upon particle size (e.g., the nominal particlediameter assuming a roughly spherical particle geometry), particledensity, the initial velocity at which the particle impacts the skinsurface, and the density and kinematic viscosity of the skin. In thisregard, optimal particle densities for use in needleless injectiongenerally range between about 0.1 and 25 g/cm³, preferably between about0.8 and 1.5 g/cm³, and injection velocities generally range betweenabout 150 and 3,000 m/sec.

A particularly unique feature of the needleless syringe is the abilityto optimize the depth of penetration of delivered particles, therebyallowing for targeted administration of pharmaceuticals to varioussites. For example, particle characteristics and/or device operatingparameters can be selected to provide for penetration depths for, interalia, epidermal or dermal delivery. One approach entails the selectionof particle size, particle density and initial velocity to provide amomentum density (e.g., particle momentum divided by particle frontalarea) of between about 2 and 10 kg/sec/m, and more preferably betweenabout 4 and 7 kg/sec/m. Such control over momentum density allows fortissue-selective delivery of the pharmaceutical particles.

Accordingly, there is a need to provide a reliable method for preparingsufficiently dense particles (having a density of about 0.8 to 1.5g/cm³) which have an average size of about 0.1 to 150 μm from a widevariety of pharmaceutical compositions. These pharmaceutical particlescan thus be transdermally delivered to a subject using a needlelesssyringe system.

Needleless syringes, such as those described above, also provide aunique means for gene therapy and nucleic acid immunization. Thesetechniques provide for the transfer of a desired gene into a subjectwith the subsequent in vivo expression thereof. Gene transfer can beaccomplished by transfecting the subject's cells or tissues ex vivo andreintroducing the transformed material into the host. Alternatively,genes can be administered directly to the recipient.

A number of methods have been developed for gene delivery in thesecontexts. For example, viral-based systems using, e.g., retrovirus,adenovirus, and adeno-associated viral vectors, have been developed forgene delivery. However, these systems pose the risk of delivery ofreplication-competent viruses. Hence, nonviral methods for directtransfer of genes into recipient cells and tissues are desirable.

Nonviral methods of gene transfer often rely on mechanisms employed bymammalian cells for the uptake and intracellular transport ofmacromolecules. For example, receptor-mediated methods of gene transferhave been developed. The technique utilizes complexes between plasmidDNA and polypeptide ligands that can be recognized by cell surfacereceptors. However, data suggests that this method may permit onlytransient expression of genes and thus has only limited application.

Additionally, microinjection techniques have been developed for thedirect injection of genetic material into cells. The technique, however,is laborious and requires single cell manipulations. Thus, the method isinappropriate for use on a large scale.

Direct injection of DNA-containing solutions into the interstitial spacefor subsequent uptake by cells has also been described. For example,International Publication No. WO 90/11092, published 4 Oct. 1990,describes the delivery of isolated polynucleotides to the interior ofcells wherein the isolated polynucleotides are delivered into theinterstitial space of the tissue and then taken up by individual cellsto provide a therapeutic effect. Such methods entail the injection ofthe DNA-containing solutions into tissue using conventional needles orcannulas, and are therefore not well suited for long term therapies orfor field or home applications.

Biolistic particle delivery systems (particle bombardment systems) havealso been developed for gene delivery into plant cells. Such techniquesuse a “gene gun” to introduce DNA-coated microparticles, such asDNA-coated metals, into cells at high velocities. The coated metals(biolistic core carriers) are generally propelled into cells using anexplosive burst of an inert gas such as helium. See, e.g., U.S. Pat. No.5,100,792 to Sanford et al. The technique allows for the direct,intracellular delivery of small amounts of DNA.

Biolistic core carriers upon which the DNA is coated, such as tungsten,gold, platinum, ferrite, polystyrene or latex, have to date been neededto achieve adequate gene transfer frequency by such direct injectiontechniques. See, e.g., International Publication No. WO 94/23738,published Oct. 27, 1994. In particular, these materials have beenselected based on their availability in defined particle sizes around 1μm in diameter, as well as providing a sufficiently high density toachieve the momentum required for cell wall or cell membranepenetration. Additionally, common biolistic core carriers are chemicallyinert to reduce the likelihood of explosive oxidation of finemicroprojectile powders, are non-reactive with DNA and other componentsof the precipitating mixes, and display low toxicity to target cells.See e.g., Particle Bombardment Technology for Gene Transfer, (1994)Yang, N. ed., Oxford University Press, New York, N.Y. pages 10-11.

However, such biolistic techniques are not appropriate for use withlarge DNA molecules since precipitation of such molecules onto corecarriers can lead to unstable configurations which will not withstandthe shear forces of gene gun delivery.

Accordingly, there remains a need to provide a highly efficient methodfor introducing therapeutically relevant DNA or other nucleic acidmolecules into mammalian tissue cells wherein the method avoids theproblems commonly encountered with prior gene delivery techniques.

DISCLOSURE OF THE INVENTION

The present invention is based on the surprising discovery thatsubstantially solid particles of nucleic acid molecules having a nominalaverage diameter of at least about 0.1 μm, preferably at least about 10μm (which are therefore larger than the average mammalian cell), can bedelivered into cells of mammalian tissue without the need for biolisticcore carriers. The result is unexpected because it was heretoforebelieved that only small DNA-coated core carrier particles, having anextremely high particle density and a much smaller size than a typicalmammalian cell, could adequately be used as microprojectiles inbiolistic gene delivery techniques. See e.g., Particle BombardmentTechnology for Gene Transfer, (1994) Yang, N. ed., Oxford UniversityPress, New York, N.Y. pages 10-11.

In the practice of the invention, powdered nucleic acid molecules aredelivered using needleless injection techniques. In particular, a noveldelivery system that uses a needleless syringe to fire solid particlesof therapeutic agents in controlled doses into and through intact skinhas recently been described in commonly owned U.S. Pat. No. 5,630,796.The patent describes a needleless syringe that delivers pharmaceuticalparticles entrained in a high velocity gas flow. The needleless syringecan be used for transdermal delivery of powdered drug compounds andcompositions, for delivery of genetic material into living cells (e.g.,gene therapy) and for the delivery of biopharmaceuticals to skin,muscle, blood or lymph. The needleless syringe can also be used inconjunction with surgery to deliver drugs and biologics to organsurfaces, solid tumors and/or to surgical cavities (e.g., tumor beds orcavities after tumor resection)

Furthermore, the nucleic acids to be delivered can be converted fromnon-dense pharmaceutical powders or particulate formulations (e.g.,those having particle densities below that required for transdermaldelivery from a needleless syringe) into densified (compacted) particlesthat are optimally suited for transdermal delivery using a needlelesssyringe. The method is equally applicable to densification ofpharmaceutical agents other than nucleic acids.

Accordingly, in one embodiment, the invention is directed to a methodfor delivering solid particles comprised of nucleic acid molecules tomammalian tissue for the genetic transformation of cells in the tissuewith the delivered nucleic acids. In a substantial departure fromconventional particle bombardment techniques, the nucleic acid particlestransferred using the method of the present invention are not deliveredusing biolistic core carriers. Furthermore, the molecules can have aparticle size that is equal to or larger than the average mammalian cellsize.

More particularly, densified particles comprised of selected nucleicacid molecules and, optionally, suitable vehicles or excipients, areprepared for delivery to mammalian tissue via a needleless syringe whichis capable of expelling the particles at delivery velocities approachingMach 1 to Mach 8 speeds. The particles have an average size that is atleast about 0.1 μm, wherein an optimal particle size is usually at leastabout 10 to 15 μm (equal to or larger than the size of a typicalmammalian cell). However, nucleic acid particles having average particlesizes of 250 μm or greater can also be delivered using the presentmethod. The depth that the delivered particles will penetrate thetargeted tissue depends upon particle size (e.g., the nominal particlediameter assuming a roughly spherical particle geometry), particledensity, the initial velocity at which the particle impacts the tissuesurface, and the density and kinematic viscosity of the tissue. In thisregard, optimal individual particle densities (e.g., in contrast to bulkpowder density) for use in needleless injection generally range betweenabout 0.1 and 25 g/cm³, and injection velocities generally range betweenabout 150 and 3,000 m/sec.

In various aspects of the invention, the above method can be practicedin vivo to provide targeted delivery of the nucleic acid particles to atarget tissue, such as delivery to the epidermis (for gene therapyapplications) or to the stratum basal layer of skin (for nucleic acidimmunization applications). In these aspects of the invention, particlecharacteristics and/or device operating parameters are selected toprovide optimal tissue-specific delivery. One particular approachentails the selection of particle size, particle density and initialvelocity to provide a momentum density (e.g., particle momentum dividedby particle frontal area) of between about 2 and 10 kg/sec/m, and morepreferably between about 4 and 7 kg/sec/m. Such control over momentumdensity allows for precisely controlled, tissue-selective delivery ofthe nucleic acid particles.

In other aspects of the invention, the needleless syringe is used totransfect cells or tissues ex vivo with the particulate nucleic acidmolecules, wherein the transformed cells are subsequently reintroducedinto the host.

In another embodiment of the invention, a method is provided forconverting non-dense pharmaceutical powders or particulate formulations(e.g., having density characteristics below that required fortransdermal delivery from a needleless syringe) into densified(compacted) particles that are optimally suited for transdermal deliveryusing a needleless syringe. Such particles have an optimal particledensity ranging from about 0.1 to about 25 g/cm³, preferably rangingfrom about 0.5 to about 3.0 g/cm³, and most preferably ranging fromabout 0.8 to about 1.5 g/cm³. The densified particles are processed toobtain optimal particle sizes ranging from about 0.1 to about 250 μm,preferably ranging from about 0.1 to about 150 μm, and most preferablyranging from about 20 to about 60 μm. The method entails the compactionof a pharmaceutical composition using high pressure and, optionallyvacuum, to densify the composition. The resulting compacted material isthen size-reduced using conventional methods to provide densifiedparticles of optimized size.

In a related embodiment of the invention, a method is provided foroptimizing the density and particle size of a particulate pharmaceuticalcomposition that has particle size and density characteristics that fallwithin the above ranges. These particles are rendered more suitable forneedleless syringe delivery using the above-described compaction andsize-reduction techniques. In this manner, the penetration depths thatare obtained when the optimized particles are delivered using aneedleless syringe can be adjusted to provide targeted dermal orintra-dermal delivery.

In a further related embodiment, the invention pertains to a method foroptimizing the particle size and density of a lyophilized or spray-driedbiopharmaceutical composition. The method entails the compaction of alyophilized or spray-dried pharmaceutical powder to obtain a densifiedmaterial. The densified material can then be reground to producecompositions in which the individual particles approach the theoreticalmaximum density and are thus optimal for delivery by impact with andpenetration into the target tissue at high velocities when deliveredfrom a needleless syringe. In a particular embodiment, lyophilizedrecombinant human growth hormone (rhGH) powder is densified to obtainparticles in the range of about 20 to 50 μm and having a bulk density ofabout 0.8 to 1.5 g/cc³. The densified rhGH particles are ideally suitedfor delivery from a needleless syringe device.

In another embodiment, the invention is directed to a compactedparticulate pharmaceutical composition formed from a porouspharmaceutical preparation. The compacted composition has an averageparticle size in the range of 0.1 to 250 μm mean diameter, a particledensity in the range of 0.1 to 25 g/cm³, and a bulk density of at leastabout 0.5 g/cc³. Needleless syringes comprising the compactedparticulate pharmaceutical preparation, as well as single-dosecontainers for a needleless syringe comprising the same, are alsoprovided.

In another embodiment, the invention is directed to a method ofdelivering a selected pharmaceutical agent to a vertebrate subject. Themethod comprises providing a densified (compacted) particulatepharmaceutical preparation as described above and delivering thepreparation to a target tissue of the vertebrate subject by needlelesssyringe.

In yet a further embodiment, the invention is directed to particles of asuitable size and density for transdermal delivery by needlelessinjection, consisting of a gene construct and an excipient selected fromthe group consisting of pharmaceutical grades of dextrose, sucrose,lactose, trehalose, mannitol, sorbitol, inositol, erythritol, dextrans,cyclodextrans, starch, cellulose, sodium or calcium phosphates, calciumsulfates, citric acid, tartaric acid, glycine, albumin, gelatin,polyacrylates, high molecular weight polyethylene glycols, andcombinations thereof.

These and other embodiments of the subject invention will readily occurto those of skill in the art in light of the disclosure herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a pictorial representation of an ex vivo delivery apparatushaving a needleless syringe arranged over a tissue culture platecontaining cells to be transformed with the particulate nucleic acidpreparations described herein.

FIG. 2 is a histogram depicting transformation efficiencies obtainedusing the apparatus of FIG. 1 to deliver DNA particles at 30 barpressure over a 60 mm target distance as described in Example 1. In theFigure, “B/P” refers to transformation efficiency (expressed as thenumber of blue cells/per dish), “F#2” and “F#3” refer to preparation 2and preparation 3, respectively, “TCC” refers to the contemporaneousdelivery of DNA-coated tungsten particles, and “THC” refers to ahistorical delivery of DNA-coated tungsten particles.

FIG. 3 is a graph depicting the transformation efficiencies obtainedusing the apparatus of FIG. 1 to deliver DNA particles at 30 barpressure over a range of target distances, also as described inExample 1. In the Figure, “B/P” refers to transformation efficiency(expressed as the number of blue cells/per culture dish), and “d(mm)”refers to target distance expressed in mm.

FIG. 4 depicts a comparison of the mean in vivo serum levels ofrecombinant human growth hormone (rhGH) in animals that wereadministered lyophilized rhGH powder by needleless injection (▴),densified rhGH particles (prepared by the method of the invention) byneedleless injection (▪), or lyophilized rhGH powder by sub-cutaneousinjection (●)

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of molecular biology and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, et al., Molecular Cloning:A Laboratory Manual (2nd Edition, 1989); Maniatis et al., MolecularCloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach,vol. I & II (D. Glover, ed.); Perbal, A Practical Guide to MolecularCloning.

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particularpharmaceutical formulations or process parameters as such may, ofcourse, vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments of theinvention only, and is not intended to be limiting.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the content clearly dictates otherwise. Thus, for example,reference to “a nucleic acid molecule” includes a mixture of two or morenucleic acid molecules, reference to “an excipient” includes mixtures oftwo or more excipients, and the like.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

A. DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. The following terms areintended to be defined as indicated below.

The term “transdermal” delivery captures both transdermal (or“percutaneous”) and transmucosal administration, i.e., delivery bypassage of a drug or pharmaceutical agent through the skin or mucosaltissue. See, e.g., Transdermal Drug Delivery: Developmental Issues andResearch Initiatives, Hadgraft and Guy (eds.), Marcel Dekker, Inc.,(1989); Controlled Drug Delivery: Fundamentals and Applications,Robinson and Lee (eds.), Marcel Dekker Inc., (1987); and TransdermalDelivery of Drugs, Vols. 1-3, Kydonieus and Berner (eds.), CRC Press,(1987). Aspects of the invention which are described herein in thecontext of “transdermal” delivery, unless otherwise specified, are meantto apply to both transdermal and transmucosal delivery. That is, thecompositions, systems, and methods of the invention, unless explicitlystated otherwise, should be presumed to be equally applicable totransdermal and transmucosal modes of delivery.

By “needleless syringe” is meant an instrument which delivers aparticulate composition transdermally, without a conventional needlethat pierces the skin. Needleless syringes for use with the presentinvention are discussed throughout this document.

As used herein, the term “drug” or “pharmaceutical agent” intends anycompound or composition of matter which, when administered to anorganism (human or animal) induces a desired pharmacologic and/orphysiologic effect by local and/or systemic action. The term thereforeencompasses those compounds or chemicals traditionally regarded asdrugs, vaccines, as well as biopharmaceuticals including molecules suchas peptides, hormones, nucleic acids, gene constructs and the like. Moreparticularly, the term “drug” or “pharmaceutical agent” includescompounds or compositions for use in all of the major therapeutic areasincluding, but not limited to, anti-infectives such as antibiotics andantiviral agents; analgesics and analgesic combinations; local andgeneral anesthetics; anorexics; antiarthritics; antiasthmatic agents;anticonvulsants; antidepressants; antihistamines; anti-inflammatoryagents; antinauseants; antineoplastics; antipruritics; antipsychotics;antipyretics; antispasmodics; cardiovascular preparations (includingcalcium channel blockers, beta-blockers, beta-agonists andantiarrythmics); antihypertensives; diuretics; vasodilators; centralnervous system stimulants; cough and cold preparations; decongestants;diagnostics; hormones; bone growth stimulants and bone resorptioninhibitors; immunosuppressives; muscle relaxants; psychostimulants;sedatives; tranquilizers; proteins peptides and fragments thereof(whether naturally occurring, chemically synthesized or recombinantlyproduced); and nucleic acid molecules (polymeric forms of two or morenucleotides, either ribonucleotides (RNA) or deoxyribonucleotides (DNA)including both double- and single-stranded molecules, gene constructs,expression vectors, antisense molecules and the like).

The above drugs or pharmaceutical agents, alone or in combination withother drugs or agents, are typically prepared as pharmaceuticalcompositions which can contain one or more added materials such ascarriers, vehicles and/or excipients. The terms “carriers,” “vehicles”and “excipients” are used interchangeably herein and generally refer tosubstantially inert materials which are nontoxic and do not interactwith other components of the composition in a deleterious manner.However, these terms do not encompass biolistic core carriers. The termscapture materials that can be used to increase the amount of solids inparticulate pharmaceutical compositions, such as those prepared usingspray-drying or lyophilization techniques. Examples of suitable carriersinclude water, silicone, gelatin, waxes, and like materials. Examples ofnormally employed “excipients,” include pharmaceutical grades ofdextrose, sucrose, lactose, trehalose, erythritol, dextrans,cyclodextrans mannitol, sorbitol, inositol, starch, cellulose, sodium orcalcium phosphates, calcium sulfate, citric acid, tartaric acid,glycine, albumin, gelatin, polyacrylates high molecular weightpolyethylene glycols (PEG), and combinations thereof.

“Gene delivery” refers to methods or systems for reliably insertingforeign DNA into host cells. Such methods can result in expression ofnon-integrated transferred DNA, extrachromosomal replication andexpression of transferred replicons (e.g., episomes), or integration oftransferred genetic material into the genomic DNA of host cells.

The nucleotide sequences are generally present in a suitable nucleicacid molecule and delivered in the form of vectors. By “vector” is meantany genetic element, such as a plasmid, phage, transposon, cosmid,chromosome, virus, virion, etc., which is capable of replication whenassociated with the proper control elements and which can transfer genesequences between cells.

A “nucleotide sequence” or a “nucleic acid molecule” refers to DNA andRNA sequences. The term captures molecules that include any of the knownbase analogues of DNA and RNA such as, but not limited to4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine,pseudoisocytosine, 5-(carboxyhydroxylmethyl)uracil, 5-fluorouracil,5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methyl-cytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycaronylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine.

A “coding sequence” or a sequence which “encodes” a particularpolypeptide, is a nucleic acid sequence which is transcribed (in thecase of DNA) and translated (in the case of mRNA) into a polypeptide invitro or in vivo when placed under the control of appropriate regulatorysequences. The boundaries of the coding sequence are conventionallydetermined by a start codon at the 5′ (amino) terminus and a translationstop codon at the 3′ (carboxy) terminus. A coding sequence can include,but is not limited to, cDNA from procaryotic or eukaryotic mRNA, genomicDNA sequences from procaryotic or eukaryotic DNA, and even synthetic DNAsequences. A transcription termination sequence will usually be located3′ to the coding sequence.

The term DNA “control sequences” refers collectively to promotersequences, polyadenylation signals, transcription termination sequences,upstream regulatory domains, origins of replication, internal ribosomeentry sites (“IRES”), enhancers, and the like, which collectivelyprovide for the replication, transcription and translation of a codingsequence in a recipient cell. Not all of these control sequences needalways be present so long as the selected gene is capable of beingreplicated, transcribed and translated in an appropriate recipient cell.

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their usualfunction. Thus, control sequences operably linked to a coding sequenceare capable of effecting the expression of the coding sequence. Thecontrol sequences need not be contiguous with the coding sequence, solong as they function to direct the expression thereof. Thus, forexample, intervening untranslated yet transcribed sequences can bepresent between a promoter sequence and the coding sequence and thepromoter sequence can still be considered “operably linked” to thecoding sequence.

By “isolated” when referring to a nucleotide sequence, or a nucleic acidmolecule containing the nucleotide sequence, is meant that the indicatedmolecule is present in the substantial absence of other biologicalmacromolecules of the same type. Thus, an “isolated nucleic acidmolecule which encodes a particular polypeptide” refers to a nucleicacid molecule which is substantially free of other nucleic acidmolecules that no not encode the subject polypeptide; however, themolecule may include some additional bases or moieties which do notdeleteriously affect the basic characteristics of the composition.

The term “transfection” is used to refer to the uptake of foreign DNA bya host cell, and a host cell has been “transformed” as a result ofhaving been transfected. The foreign DNA may or may not be integrated(covalently linked) to chromosomal DNA making up the genome of the cell.By “host cell,” or “host mammalian cell” is meant a cell which has beentransfected, or is capable of being transfected, by a nucleic acidmolecule containing a nucleotide sequence of interest. The term includesthe progeny of the parent cell, whether or not the progeny is identicalin morphology or in genetic make-up to the original parent, so long asthe nucleotide sequence of interest is present within the cell.

By “biolistic core carrier” is meant a carrier on which a nucleic acid(e.g., DNA) is coated in order to impart a defined particle size as wellas a sufficiently high density to achieve the momentum required for cellwall penetration, such that the DNA can be delivered using biolistictechniques, such as by use of a gene gun (see, e.g., U.S. Pat. No.5,100,792). Biolistic core carriers typically include dense solids suchas tungsten, gold, platinum, ferrite, polystyrene and latex. See e.g.,Particle Bombardment Technology for Gene Transfer, (1994) Yang, N. ed.,Oxford University Press, New York, N.Y. pages 10-11.

By “vertebrate subject” is meant any member of the subphylum cordata,particularly mammals, including, without limitation, humans and otherprimates. The term does not denote a particular age. Thus, both adultand newborn individuals are intended to be covered.

B. MODES OF CARRYING OUT THE INVENTION

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular formulationsor process parameters as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments of the invention only, and is notintended to be limiting.

Although a number of methods and materials similar or equivalent tothose described herein can be used in the practice of the presentinvention, the preferred materials and methods are described herein.

As explained above, the present invention allows for the highlyefficient delivery of solid particles of nucleic acid molecules having anominal average diameter of at least about 0.1 μm, preferably at leastabout 10 μm, to mammalian tissues. The method utilizes biolistic genetransfer techniques yet surprisingly allows for the delivery of nucleicacid molecules without the need for biolistic core carriers.

A wide variety of nucleic acid molecules can be delivered using themethods of the invention. Generally, the molecules contain codingregions with suitable control sequences or other therapeuticallyrelevant nucleotide sequences. The nucleic acid molecules are preparedin the form of vectors which include the necessary elements to directtranscription and translation in a host cell. If expression is desiredusing the host's enzymes (such as by the use of endogenous RNApolymerase) , the gene or genes will be present in the vectorsoperatively linked to control sequences recognized by the particularhost, or even particular cells within the host. Thus, eucaryotic andphage control elements will be present for expression in mammalianhosts. Such sequences are known in the art and are discussed more fullybelow.

Suitable nucleotide sequences for use in the delivery methods of thepresent invention include any therapeutically relevant nucleotidesequence. Thus, the present invention can be used to deliver one or moregenes encoding a protein defective or missing from a target cell genomeor one or more genes that encode a non-native protein having a desiredbiological or therapeutic effect (e.g., an antiviral function). Theinvention can also be used to deliver a nucleotide sequence capable ofproviding immunity, for example an immunogenic sequence that serves toelicit a humoral and/or cellular response in a subject, or a sequencethat corresponds to a molecule having an antisense or ribozyme function.

Suitable genes which can be delivered include those used for thetreatment of inflammatory diseases, autoimmune, chronic and infectiousdiseases, including such disorders as AIDS, cancer, neurologicaldiseases, cardiovascular disease, hypercholestemia; various blooddisorders including various anemias, thalassemia and hemophilia; geneticdefects such as cystic fibrosis, Gaucher's Disease, adenosine deaminase(ADA) deficiency, emphysema, etc. A number of antisense oligonucleotides(e.g., short oligonucleotides complementary to sequences around thetranslational initiation site (AUG codon) of an mRNA) that are useful inantisense therapy for cancer and for viral diseases have been describedin the art. See, e.g., Han et al. (1991) Proc. Natl. Acad. Sci. USA88:4313; Uhlmann et al. (1990) Chem. Rev. 90:543; Helene et al. (1990)Biochim. Biophys. Acta. 1049:99 Agarwal et al. (1988) Proc. Natl. Acad.Sci. USA 85:7079; and Heikkila et al. (1987) Nature 328:445. A number ofribozymes suitable for use herein have also been described. See, e.g.,Cech et al. (1992) J. Biol. Chem. 267:17479 and U.S. Pat. No. 5,225,347to Goldberg et al.

For example, in methods for the treatment of solid tumors, genesencoding toxic peptides (e.g., chemotherapeutic agents such as ricin,diphtheria toxin and cobra venom factor), tumor suppressor genes such asp53, genes coding for mRNA sequences which are antisense to transformingoncogenes, antineoplastic peptides such as tumor necrosis factor (TNF)and other cytokines, or transdominant negative mutants of transformingoncogenes, can be delivered for expression at or near the tumor site.

Similarly, genes coding for peptides known to display antiviral and/orantibacterial activity, or stimulate the host's immune system, can alsobe administered. Thus, genes encoding many of the various cytokines (orfunctional fragments thereof), such as the interleukins, interferons,and colony stimulating factors, will find use with the instantinvention. The gene sequences for a number of these substances areknown.

For the treatment of genetic disorders, functional genes correspondingto genes known to be deficient in the particular disorder can beadministered to the subject. The instant methods will also find use inantisense therapy, e.g., for the delivery of oligonucleotides able tohybridize to specific complementary sequences thereby inhibiting thetranscription and/or translation of these sequences. Thus, DNA or RNAcoding for proteins necessary for the progress of a particular diseasecan be targeted, thereby disrupting the disease process. Antisensetherapy, and numerous oligonucleotides which are capable of bindingspecifically and predictably to certain nucleic acid target sequences inorder to inhibit or modulate the expression of disease-causing genes areknown and readily available to the skilled practitioner. Uhlmann et al.(1990) Chem. Rev. 90:343, Neckers et al (1992) Crit. Rev. Oncogenesis3:175; Simons et al. (1992) Nature 359:67; Bayever et al. (1992)Antisense Res. Dev. 2:109; Whitesell et al. (1991) Antisense Res. Dev.1:343; Cook et al. (1991) Anti-Cancer Drug Design 6:58S; Eguchi et al.(1991) Annu. Rev. Biochem. 60:631. Accordingly, antisenseoligonucleotides capable of selectively binding to target sequences inhost cells are provided herein for use in antisense therapeutics.

For nucleic acid immunizations, antigen-encoding expression vectors canbe delivered to a subject for the purpose of eliciting humoral and/orcellular immune responses to antigens encoded by the vector. Inparticular, humoral, cytotoxic cellular and protective immune responseselicited by direct intramuscular injection of antigen-encoding DNAs havebeen described. Tang et al. (1992) Nature 358:152; Davis et al. (1993)Hum. Molec. Genet. 2:1847; Ulmer et al. (1993) Science 258:1745; Wang etal. (1993) Proc. Natl. Acad. Sci. USA 90:4156; Eisenbraun et al. (1993)DNA Cell Biol. 12:791; Fynan et al. (1993) Proc. Natl. Acad. Sci. USA90:12476; Fuller et al. (1994) AIDS Res. Human Retrovir. 10:1433; andRaz et al. (1994) Proc. Natl. Acad. Sci. USA 91:9519. In addition, theseimmune responses have also been elicited using biolistic techniques.See, e.g., EP 0500799.

Isolation of Genes and Construction of Vectors:

Nucleotide sequences selected for use in the present invention can bederived from known sources, for example, by isolating the same fromcells containing a desired gene or nucleotide sequence using standardtechniques. Similarly, the nucleotide sequences can be generatedsynthetically using standard modes of polynucleotide synthesis that arewell known in the art. See, e.g., Edge et al. (1981) Nature 292:756;Nambair et al. (1984) Science 223:1299; Jay et al. (1984) J. Biol. Chem.259:6311. Generally, synthetic oligonucleotides can be prepared byeither the phosphotriester method as described by Edge et al. (supra)and Duckworth et al. (1981) Nucleic Acids Res. 9:1691, or thephosphoramidite method as described by Beaucage et al. (1981) Tet.Letts. 22:1859, and Matteucci et al. (1981) J. Am. Chem. Soc. 103:3185.Synthetic oligonucleotides can also be prepared using commerciallyavailable automated oligonucleotide synthesizers. The nucleotidesequences can thus be designed with appropriate codons for a particularamino acid sequence. In general, one will select preferred codons forexpression in the intended host. The complete sequence is assembled fromoverlapping oligonucleotides prepared by standard methods and assembledinto a complete coding sequence. See, e.g., Edge et al. (supra); Nambairet al. (supra) and Jay et al. (supra).

A particularly convenient method for obtaining nucleic acid sequencesfor use herein is by recombinant means. Thus, a desired nucleotidesequence can be excised from a plasmid carrying the same using standardrestriction enzymes and procedures. Site specific DNA cleavage isperformed by treating with the suitable restriction enzyme (or enzymes)under conditions which are generally understood in the art, and theparticulars of which are specified by manufacturers of commerciallyavailable restriction enzymes. If desired, size separation of thecleaved fragments may be performed by polyacrylamide gel or agarose gelelectrophoresis using standard techniques.

Restriction cleaved fragments may be blunt ended by treating with thelarge fragment of E. coli DNA polymerase I (Klenow) in the presence ofthe four deoxynucleotide triphosphates (dNTPs) using standardtechniques. The Klenow fragment fills in at 5′ single-stranded overhangsbut digests protruding 3′ single strands, even though the four dNTPs arepresent. If desired, selective repair can be performed by supplying onlyone, or several, selected dNTPs within the limitations dictated by thenature of the overhang. After Klenow treatment, the mixture can beextracted with e.g. phenol/chloroform, and ethanol precipitated.Treatment under appropriate conditions with 51 nuclease or BAL-31results in hydrolysis of any single-stranded portion.

PCR techniques can also be used in order to obtain a nucleic acidmolecule of interest. Generally, the technique involves amplification ofsequences from a human genomic or cDNA library. Degenerate ornondegenerate oligonucleotide primers for PCR may be prepared based onknown amino acid sequences or on sequences of homologous genes. Theproducts of such PCR reactions may be selected according to size by gelelectrophoresis. Such PCR methods are described in e.g., U.S. Pat. Nos.4,965,188; 4,800,159; 4,683,202; 4,683,195.

Once coding sequences for desired peptides or proteins have beenprepared or isolated, such sequences can be cloned into any suitablevector or replicon. Numerous cloning vectors are known to those of skillin the art, and the selection of an appropriate cloning vector is amatter of choice. Ligations to other sequences are performed usingstandard procedures, known in the art.

Selected nucleotide sequences can be placed under the control ofregulatory sequences such as a promoter, ribosome binding site and,optionally, an operator (collectively referred to herein as “control”elements), so that the sequence encoding the desired protein istranscribed into RNA in the host tissue transformed by a vectorcontaining this expression construct. The coding sequence may or may notcontain a signal peptide or leader sequence.

The choice of control elements will depend on the host being transformedand the type of preparation used. Thus, if the host's endogenoustranscription and translation machinery will be used to express theproteins, control elements compatible with the particular host will beutilized. In this regard, several promoters for use in mammalian systemsare known in the art and include, but are not limited to, promotersderived from SV40, CMV, HSV, RSV, MMTV, T7, T3, among others. Similarly,promoters useful with procaryotic enzymes are known and include the tac,spa, trp, trp-lac λ-p_(L), T7, phoA promoters, as well as others.

In addition to control sequences, it may be desirable to add regulatorysequences which allow for regulation of the expression of proteinsequences encoded by the delivered nucleotide sequences. Regulatorysequences are known to those of skill in the art, and examples includethose which cause the expression of a coding sequence to be turned on oroff in response to a chemical or physical stimulus, including thepresence of a regulatory compound. Other types of regulatory elementsmay also be present in the vector, for example, enhancer sequences.

An expression vector is constructed so that the particular codingsequence is located in the vector with the appropriate control and,optionally, regulatory sequences such that the positioning andorientation of the coding sequence with respect to the control sequencesallows the coding sequence to be transcribed under the “control” of thecontrol sequences (i.e., RNA polymerase which binds to the DNA moleculeat the control sequences transcribes the coding sequence). Modificationof the sequences encoding the particular protein of interest may bedesirable to achieve this end. For example, in some cases it may benecessary to modify the sequence so that it is attached to the controlsequences with the appropriate orientation; i.e., to maintain thereading frame. The control sequences and other regulatory sequences maybe ligated to the coding sequence prior to insertion into a vector.Alternatively, the coding sequence can be cloned directly into anexpression vector which already contains the control sequences and anappropriate restriction site.

Preparation of Particulate Molecules:

Once obtained and/or constructed, the nucleic acid molecules areprepared for delivery in particulate form. For example, particulatemolecules can be produced using particle formation techniques well knownin the art, such as but not limited to spray-drying, spray-coating,freeze-drying (lyophilization) and super critical fluid precipitation.

In one embodiment, the invention entails a procedure for forming denseparticles from low density particulate pharmaceutical preparations. Inparticular, manufacturing processes for preparing pharmaceuticalparticles from delicate molecules such as DNA, proteins or peptidesgenerally result in low density particles having either a hollowspherical or open lattice monolithic structure. Such particles arepoorly suited for use in needleless syringe delivery systems, whereinthe particles must have sufficient physical strength to withstand suddenacceleration to velocities approaching the speed of sound and the impactwith, and passage through, the skin and tissue.

One common method of preparing particulate biopharmaceuticals, such asnucleic acids, is lyophilization (freeze-drying). Lyophilization relatesto a technique for removing moisture from a material and involves rapidfreezing at a very low temperature, followed by rapid dehydration bysublimation in a high vacuum. This technique typically yieldslow-density porous particles having an open matrix structure. Suchparticles are chemically stable, but are rapidly reconstituted(disintegrated and/or brought into solution) when introduced into anaqueous environment.

Another method of providing particulate preparations that can be usedwith these and other delicate or heat-sensitive biomolecules isspray-drying. Spray-drying relates to the atomization of a solution ofone or more solids using a nozzle, spinning disk or other device,followed by evaporation of the solvent from the droplets. Moreparticularly, spray-drying involves combining a highly dispersed liquidpreparation (e.g., a solution, slurry, emulsion or the like) with asuitable volume of hot air to produce evaporation and drying of theliquid droplets. Spray-dried pharmaceuticals are generally characterizedas homogenous spherical particles that are frequently hollow. Suchparticles have low density and exhibit a rapid rate of solution.

The low-density particulate solids produced by lyophilization andspray-drying techniques are ideal for redissolution for parenteraladministration in solution via syringe or catheters. However, suchparticles are not useful for delivery from a needleless syringe in asolid form. Accordingly, for purposes of the present method, thepreparations are densified to provide particles including nucleic acidmolecules that are much better suited for delivery using a needlelesssyringe (e.g., substantially solid particles having a size of about 50μm and a density of at least about 0.9 to 1.5 g/cc³). In particular, theopen lattice or hollow shell particles provided by spray-drying orlyophilization can be condensed without heating or shear to providedense materials that can be milled or otherwise size-reduced to yieldpharmaceutical particles having both size and density characteristicssuitable for delivery by needleless injection.

The nucleic acids for delivery by the method of the invention, may beinitially prepared in a formulation suitable for spray-drying orlyophilization. Such formulations generally require only a solution inwhich the nucleic acids will be stable for freezing and lyophilizationand, optionally, an excipient for the drying procedure which isacceptable for parenteral delivery. In this regard, suitable excipientsmay be added to the formulations to provide sufficient mass for anindividual dose, enabling measurement of doses by practical processes,e.g., by weight or volume. Typical dosages can be about 0.5 to about 5mg, preferably about 1 to about 2 mg. Suitable excipients include, butare not limited to, carbohydrates (such as trehalose, glucose, dextroseand sucrose) or polyols (such as mannitol). Amino acids such as glycineand its hydrochloride salt can be used as buffers as well as phosphate,lactate or citrate buffers, among others. Additionally, any knowncomposition for DNA stabilization will find use in the presentformulations. The compositions may optionally include additive agentssuch as cryoprotectants, antioxidants, or the like. Adjustingcompositions to enhance physical and chemical stability of the variousparticulate nucleic acid formulations provided herein is within theordinary skill in the art.

One particular approach to stabilization during reprocessing of thenucleic acid formulations entails the use of additives which arecombined with the solution prior to freezing for lyophilization to causethe nucleic acids to coil or ball and thus provide the genetic materialas a discontinuous phase in the otherwise microscopically homogeneousparticles. In such formulations, the bulking agent would be thecontinuous phase in the dried solid so that any grinding prior tocompression, compression densification and regrinding (as described indetail below), and any particle attrition during sizing via sieve or airclassification, acceleration and injection, would be less likely todisrupt the long chain nucleic acids. Homogeneity of the particles withrespect to nucleic acid content is critical because of the potential forsegregation by size during storage or injection.

Condensing the nucleic acid powders can be conducted by compaction in asuitable press (e.g., a hydraulic press, tableting press or rotarypress), wherein the powders are compressed at about 1,000 to 24,000pounds/square inch (e.g., 0.5 to 12 tons/square inch or 7 to 170 MPa)for a suitable time. Compaction can be carried out under vacuum ifdesired. The resulting compacted material is then coarsely regrounduntil visually broken up. The particle size is then reduced to about a20 to 50 μm average size with an optimal bulk particle density of around0.9 to 1.5 g/cm³, or as close to absolute or theoretical density aspossible. Particle size reduction can be conducted using methods wellknown in the art including, but not limited to, roller milling, ballmilling, hammer, air or impact milling, attrition milling, sieving,sonicating, or combinations thereof. The compression parameters andparticle sizing will, of course, vary depending upon the startingmaterial used, the desired target particle size and density, and likeconsiderations.

Particle density can be ascertained using helium pycnometry to treasureabsolute density and various techniques to establish porosity such asmercury intrusion BET, flotation in a density gradient, and the like.These techniques are all well known in the art.

Thus, the method can be used to obtain nucleic acid particles having asize ranging from about 10 to about 250 μm, preferably about 10 to about150 μm, and most preferably about 20 to about 60 μm; and a particledensity ranging from about 0.1 to about 25 g/cm³, and a bulk density ofabout 0.5 to about 3.0 g/cm³, or greater.

A particularly preferred method for providing nucleic acid moleculessuitable for biolistic delivery is the novel densification/compactiontechnique described herein. The technique is useful not only forpreparing nucleic acid biopharmaceuticals, but can be used to preparealmost any desired physiologically active composition for needlelessdelivery.

Accordingly, in another embodiment of the invention, a method isprovided for densifying (compacting) a non-nucleic acid pharmaceuticalpreparation.

As explained above, current manufacturing processes for preparingpharmaceutical particles from delicate molecules such as proteins orpeptides are poorly suited for use in needleless syringe deliverysystems. For example, as discussed above, lyophilization typicallyyields low-density porous particles having an open matrix structure.Exemplary biopharmaceuticals available as lyophilized particles includerecombinant human growth factor (e.g., Genotropin®, Pharmacia,Piscataway, N.J.); somatrem (e.g., Protropin®, Genentech, S. SanFrancisco, Calif.); somatropin (e.g., Humatrope®, Eli Lilly,Indianapolis, Ind.); recombinant interferon α-2a (e.g., Roferon®,Hoffman-La Roche, Nutley, N.J.) recombinant interferon α-2b (e.g.,Intron A®, Schering-Plough, Madison, N.J.); and recombinant alteplase(e.g., Activase®, Genentech, S. San Francisco, Calif.).

In addition, spray-dried pharmaceuticals are generally characterized ashomogenous spherical particles that are frequently hollow. Suchparticles have low density and exhibit a rapid rate of solution.Exemplary heat-sensitive pharmaceuticals that are prepared usingspray-drying techniques include the amino acids; antibiotics such asaureomycin, bacitracin, penicillin and streptomycin; ascorbic acid;cascara extracts; pepsin and similar enzymes; protein hydrolysates; andthiamine.

When spray-dried and lyophilized pharmaceutical particles are ground ormilled, they yield very small, light and non-dense particles that arepoorly suited for delivery through skin or mucosal tissues. Inparticular, such particles, when delivered from a needleless syringe,are often too light to have the momentum necessary to penetrate intactskin (e.g., pass through the stratum corneum) and would thus fail toenter the systemic circulation. In this regard, the stratum corneum is athin layer of dense packed, highly keratinized cells, generally about10-15 μm thick and which covers most of the human body. The stratumcorneum thus provides the primary skin barrier which atransdermally-delivered particle must cross.

Accordingly, the present method entails densifying (compacting) suchpreparations to provide particles that are much better suited fordelivery from a needleless syringe (e.g., substantially solid particleshaving a size of about 50 μm and a bulk density of at least about 0.5 to1.5 g/cc³). In particular, open lattice or hollow shell particlesprovided by spray-drying or lyophilization can be condensed withoutheating or shear to provide dense, compacted materials that can bemilled or otherwise size-reduced to yield pharmaceutical particleshaving both size and density characteristics suitable for delivery byneedleless injection.

Condensing of the spray-dried or lyophilized powders is typicallyconducted by compaction in a suitable press (e.g., a hydraulic press,tableting press or rotary press), wherein the powders are compressed atabout 1,000 to 24,000 pounds/square inch (0.5 to 12 tons/square inch)for a suitable time. This compaction can be carried out under vacuum ifdesired. The resulting compacted material is then coarsely regrounduntil visually broken up. The particle size is then reduced to about a20 to 50 μm average size to yield a bulk density of around 0.5 to 1.5g/cc³ (with a particle density of about 0.1 to 25 g/cm³). Particle sizereduction can be conducted using methods well known in the artincluding, but not limited to, roller milling, ball milling, hammer orimpact milling, attrition milling, sieving, sonicating, or combinationsthereof. The compression parameters and particle sizing will, of course,vary depending upon the starting material used, the desired targetparticle size and density, and like considerations. The startingmaterial can be any pharmaceutical preparation having a particle sizeand density which one is desirous of changing to obtain more optimalsize and density characteristics for use in needleless syringes.

Following densification, particles of suitable size can be selected andclassified using standard techniques, known in the art, such as byvibratory, sonic or jet sieving, cyclone separation, or like techniques,well known in the art.

Actual particle density, or “absolute density,” can be readilyascertained using known quantification techniques such as heliumpycnometry and the like.

Alternatively, envelope density measurements can be used to assesssuitable densification of the particulate pharmaceutical compositions.Envelope density information is useful in characterizing the density ofporous objects of irregular size and shape. Envelope density, or “bulkdensity,” is the mass of an object divided by its volume, where thevolume includes that of its pores and small cavities. A number ofmethods of determining envelope density are known in the art, includingwax immersion, mercury displacement, water absorption and apparentspecific gravity techniques. A number of suitable devices are alsoavailable for determining envelope density, for example, the Geopyc™Model 1360, available from the Micromeritics Instrument Corp. Thedifference between the absolute density and envelope density of a samplepharmaceutical composition provides information about the sample'spercentage total porosity and specific pore volume. In the practice ofthe invention, compaction of porous particulate pharmaceuticalcompositions will generally result in a reduction of porosity, and aconcomitant increase in bulk (envelope) density.

Thus, the method can be used to obtain particles having a size rangingfrom about 0.1 to about 250 μm, preferably about 0.1 to about 150 μm,and most preferably about 20 to about 60 μm; a particle density rangingfrom about 0.1 to about 25 g/cm³, and a bulk density of preferably about0.5 to about 3.0 g/cm³, and most preferably about 0.8 to about 1.5g/cm³.

The above-described method can also be used to optimize the density andparticle size of a particulate pharmaceutical composition that hasparticle size and density characteristics that fall within the aboveranges. In this manner, the penetration depths that are obtained whenthe optimized particles are delivered at high velocities using aneedleless syringe can be adjusted to optimize targeted dermal orintra-dermal delivery.

However, as noted hereinabove, the invention is particularly suited forpreparing densified particles having optimized density fromheat-sensitive biopharmaceutical preparations of peptides, polypeptides,proteins, nucleic acids and other such biological molecules. Exemplarypeptide and protein formulations which can be densified using theinstant method include, without limitation, insulin; calcitonin;octreotide; endorphin; liprecin; pituitary hormones (e.g., human growthhormone and recombinant human growth hormone (hGH and rhGH), HMG,desmopressin acetate, etc); follicle luteoids; growth factors (such asgrowth factor releasing factor (GFRF), somatostatin, somatotropin andplatelet-derived growth factor); asparaginase; chorionic gonadotropin;corticotropin (ACTH); erythropoietin (EPO); epoprostenol (plateletaggregation inhibitor); glucagon; interferons; interleukins; menotropins(urofollitropin follicle-stimulating hormone (FSH) and luteinizinghormone (LH)); oxytocin; streptokinase; tissue plasminogen activator(TPA); urokinase; vasopressin; desmopressin; ACTH analogues; angiotensinII antagonists; antidiuretic hormone agonists; bradykinin antagonists;CD4 molecules; antibody molecules and antibody fragments (e.g., Fab,Fab₂, Fv and sFv molecules); IGF-1; neurotrophic factors; colonystimulating factors; parathyroid hormone and agonists; parathyroidhormone antagonists; prostaglandin antagonists; protein C; protein S;renin inhibitors; thrombolytics; tumor necrosis factor (TNF); vaccines(particularly peptide vaccines including subunit and synthetic peptidepreparations); vasopressin antagonists analogues; and α-1 antitrypsin.Exemplary nucleic acid molecules are detailed above.

Administration of the Particles:

Following formation, the particulate preparations are delivered tomammalian tissue using a needleless syringe. The compacted particles ofthe present invention can be packaged in individual unit dosages. Asused herein, a “unit dosage” intends a dosage receptacle containing atherapeutically effective amount of a pharmaceutical powder producedaccording to the methods of the present invention. The dosage receptacleis generally one which fits within a needleless syringe device to allowfor transdermal delivery from the device. Such receptacles can take theform of capsules, foil pouches, sachets, cassettes, and the like.Appropriate needleless syringes are described herein above.

Transdermal delivery from these various needleless syringeconfigurations is carried out with particles having an approximate sizethat generally ranges between 0.1 and 250 μm. However, the optimalparticle size is usually at least about 10 to 15 μm (the size of atypical cell). Particles larger than about 250 μm can also be deliveredfrom the device, with the upper limitation being the point at which thesize of the particles would cause untoward damage to the skin. Otherparticulate biopharmaceuticals, such as peptide and proteinpreparations, will generally have an approximate size of 0.1 to about250 μm, preferably about 0.1 to about 150 μm, and most preferably about20 to about 60 μm.

The actual distance which the delivered particles will penetrate dependsupon particle size (e.g., the nominal particle diameter assuming aroughly spherical particle geometry), particle density, the initialvelocity at which the particle impacts the skin surface, and the densityand kinematic viscosity of the skin. In this regard, optimal particledensities for use in needleless injection generally range between about0.1 and 25 g/cm³, and optimal bulk densities range from about 0.5 and3.0 g/cm³. Injection velocities generally range between about 100 and3,000 m/sec.

Compositions containing a therapeutically effective amount of thepowdered molecules described herein can be delivered to any suitabletarget tissue via the above-described needleless syringes. For example,the compositions can be delivered to muscle, skin, brain, lung, liver,spleen, bone marrow, thymus, heart, lymph, blood, bone cartilage,pancreas, kidney, gall bladder, stomach, intestine, testis, ovary,uterus, rectum, nervous system, eye, gland and connective tissues. Fornucleic acid molecules, delivery is preferably to, and the moleculesexpressed in, terminally differentiated cells; however, the moleculescan also be delivered to non-differentiated, or partially differentiatedcells such as stem cells of blood and skin fibroblasts.

The powdered compositions are administered to the subject to be treatedin a manner compatible with the dosage formulation, and in an amountthat will be prophylactically and/or therapeutically effective. Theamount of the composition to be delivered, generally in the range offrom 0.5 μg/kg to 100 μg/kg of nucleic acid molecule per dose, dependson the subject to be treated. Doses for other pharmaceuticals, such asphysiological active peptides and proteins, generally range from about0.1 μg to about 20 mg, preferably 10 μg to about 3 mg. The exact amountnecessary will vary depending on the age and general condition of theindividual to be treated, the severity of the condition being treated,the particular preparation delivered, the site of administration, aswell as other factors. An appropriate effective amount can be readilydetermined by one of skill in the art.

Thus, a “therapeutically effective amount” of the present particulatecompositions will be sufficient to bring about treatment or preventionof disease or condition symptoms, and will fall in a relatively broadrange that can be determined through routine trials.

C. EXPERIMENTAL

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

EXAMPLE 1

The following experiment was conducted to investigate the possibility ofusing freeze-dried DNA as an alternative to DNA-coated metal particlesin the biolistic transfer of genetic material. In particular, powderedDNA plasmids as well as DNA-coated tungsten particles as controls weredelivered ex vivo to male human fibroblast HT1080 cells using aneedleless syringe apparatus as follows.

Clone 123 is a small plasmid of ˜11 kb which contains theβ-galactosidase marker gene so that transient transformation can bemeasured with the chromogenic indicator X-Gal. Plasmids were bulked witha carbohydrate excipient, trehalose. Trehalose was selected as theexcipient because of its stabilizing properties (Colaco et al. (1992)Bio/Technology 10:1009). The trehalose was dissolved in distilled waterand filter-sterilized prior to adding the DNA to the solution. Threedifferent solutions of DNA sugar were made up with the proportions shownbelow in Table 1. TABLE 1 Preparation 1 2 3 Clone 123 800 μg 160 μg 80μg (2.7 μg/μL) (296.3 μL) (59.3 μL) (29.6 μL) Trehalose 10 mg 10 mg 10mg (100 mg/15 ml H₂O) (1.5 ml) (1.5 ml) (1.5 ml) Payload 0.1 mg 0.5 mg1.0 mg (for 8 μg DNA)

The plasmid/sugar solutions were then freeze-dried (using solid CO₂ andisopropanol to freeze the solution prior to vacuum drying), and thefreeze-dried DNA-trehalose trehalose solid milled to form microparticlesusing an agate mortar and pestle.

As a control, DNA-coated tungsten particles were made by coatingtungsten microprojectiles (19.35×10³ kb.m⁻³) of 1.014 μm median diameter(M-17, GTE/Sylvania, Towanda, Pa., USA) with forty micrograms of Clone123 plasmid DNA, giving five payloads of 8 μg DNA, using a derivative ofknown methods for coating microparticles. Potter et al. (1984) Proc.Natl. Acad. Sci. USA 81:7161, Klein et al. (1987) Nature 327:70, andWilliams et al. (1991) Proc. Natl. Acad Sci. USA 88:2726.

More particularly, prior to coating, the tungsten particles weresterilized and brought into suspension. A 50 mg sample of 1.048 μm(median diameter) tungsten microprojectiles (M-17, GTE/Sylvania,Towanda, Pa., USA) was weighed into a 1.5 cc Eppendorf tube and thensterilized in 100% ethanol (EtOH). In order to disperse themicroprojectiles (disrupt particle aggregates), the sterilized solutionwas sonicated thoroughly by contacting the outside of the Eppendorf tubewith the probe of a sonicator. The dispersed tungsten particles werecentrifuged and the supernatant removed. The tungsten particles wereresuspended in 1 cc sterile distilled water and centrifuged for twocycles, and then stored in 1 cc sterile distilled water until coating.

The plasmid DNA was absorbed to the tungsten microprojectiles by adding20 μL of the DNA (1 mg/mL) to 40 μL of the suspension of tungstenparticles prepared above. The suspension was vortexed to ensure adequatemixing of the reagents. The following reagents were then added, in theorder given, with vortexing after each addition: 253 μL CaCl₂ (2.5 M);50 μL spermidine (0.10 M, stored frozen); and 207 μL sterile distilledH₂O. The final mixture was vortexed for 10 minutes at 4° C. TheDNA-coated tungsten microprojectiles were then centrifuged at 500 G for5 minutes. After centrifugation, all supernatant was carefully removedand 100 μL of 70% EtOH added. The coated particles were againcentrifuged, all supernatant removed, and the final preparationresuspended in 30 μL of 100% EtOH.

The above method resulted in suitable quantities of DNA-coated tungstenparticles to allow for 4 to 5 deliveries by the needleless syringe. Theabove-noted reagent quantities can, of course, be varied to providedifferent loadings of DNA in accordance with known methods. The volumeand molar concentration (M) of the stock solutions used to coat tungstenwith DNA are given below in Table 2. TABLE 2 Component Quantity (μL)Tungsten particles (50 mg/mL) 40 Clone 123 (2.7 μg/μL) 14.8 CaCl₂ (2.5M) 253 Spermidine (0.1 M) 50 distilled H₂O 212.2

For transformation, 6 cm diameter culture dishes were seeded with 5×10⁵male human fibroblast HT1080 cells 24 hours before transfection. Tworeplicate dishes were prepared for each of the treatments, and twonegative control plates were also prepared.

The microparticles and the tungsten-coated particles were then deliveredto cells using a needleless syringe as described above. The syringeincluded a 4.5 mL reservoir chamber with plunger type valve, a heliumgas reservoir, a Mach 2 nozzle and 12 μm Mylar sheet hand-punched into 6mm diameter diaphragms.

In particular, mylar diaphragms were first sterilized by singly layeringbetween pieces of filter paper, stacked one atop the other, wrapped inaluminum foil and sealed completely with autoclave tape to ensure thatno water entered the filter paper/diaphragm stack during the autoclaveprocess. This was placed inside a beaker covered with aluminum foil andplaced in an autoclave chamber.

Two 12 μm Mylar diaphragms of 6 mm diameter were used in the membranecassette. One milligram and one-half milligram payloads of thefreeze-dried DNA powder were loaded onto the lower membrane in thecassette. This payload was then covered with the other pieces of thecassette and the remaining diaphragm. Only preparations 2 and 3 wereused in the experiment because of the difficulty in weighing out smallmasses accurately. Another five of the cassettes were each loaded with 5μL of the DNA/tungsten particle suspension. All the above quantitiesgave a mass of about 8 μg of DNA being delivered in each shot regardlessof particle formulation used.

Referring now to FIG. 1, a delivery apparatus 2 was assembled whichcontained a needleless syringe 4 loaded with a cassette as describedabove. The needleless syringe 4 was arranged on a ring stand 6 using astandard tube clamp 8 to hold the syringe in position relative to aculture dish 10 seeded with the HT1080 cells 12. The distance betweenthe downstream terminus 14 of the needleless syringe 4 and the cells 14in the culture dish 10 was measured to affix a target distance,generally indicated at d. In order to optimize the parameters fordelivery of the freeze-dried DNA, either the target distance d wasvaried over a constant delivery pressure, or the delivery pressure wasvaried over a constant target distance. In particular, the DNApreparations were fired from a target distance ranging from 20 to 60 mmusing helium driver gas pressures ranging from 30 to 50 bar.

After transformation, cells were incubated for 2 days, stained, and thentransient assays with X-gal were performed to determine transformationefficiencies using previously described methods. Murray, E. J. (ed)(1991) Methods in Molecular Biology: Gene Transfer and ExpressionProtocols, Vol. 7, Humana Press, Clifton, N.J. Specifically,transformation efficiency was assessed by counting the number ofblue-stained cells. The delivery parameters and transformation resultsare depicted below in Table 3. Blast effect was rated from 1 point forquite small (diameter of dead cell zone being approximately 5-8 mm) tofive points for very large (diameter of cell zone being greater than 30mm). As can be seen, transformation by the plasmid/trehalose powderpreparation of the present invention was on the same order as thatobserved for the metallic particles.

As shown in FIG. 2, optimal transformation results were seen with theparticulate plasmid/trehalose preparation when delivered using 30 barpressure at a target distance of 60 mm. More particularly, FIG. 2provides a direct comparison of the transformation efficiency obtainedby delivery of the particulate nucleic acid preparation (bothpreparations 2 and 3) with historical and contemporary deliveries ofDNA-coated tungsten particles. Referring now to FIG. 3, data obtainedfor deliveries at 30 bar are depicted in a graph which presentstransformation efficiency as a function of target distance. As can beseen, the optimal target distance for the number 2 and 3 preparations(referred to as OBS Formulation #2 and OBS Formulation #3, respectively)was not reached; however, transformation efficiency did substantiallyincrease with increased target distances. Further, when deliveries werecarried out at the maximum distance tested (60 mm), transformationefficiencies obtained with the particulate DNA formulations (#2 and #3)were appreciably better than those observed with the DNA-coated tungstencontrols. TABLE 3 Target Blue Cell Blast Shot Formulation DistancePressure Count effect A1 Tungsten 60 30 224 2 A2 Tungsten 60 30 596 2 A3Tungsten 60 30 575 2 A4 Tungsten 60 30 581 2 A5 Tungsten 60 30 — — B1 #3trehalose 20 30 155 3 B2 #3 trehalose 20 30 227 3 C1 #3 trehalose 40 30654 2 C2 #3 trehalose 40 30 643 2 D1 #3 trehalose 40 50 394 4 D2 #3trehalose 40 50 175 5 E1 #3 trehalose 60 30 1416 1 E2 #3 trehalose 60 301654 1 F1 #3 trehalose 60 50 408 3 F2 #3 trehalose 60 50 486 3 G1 #2trehalose 20 30 166 3 G2 #2 trehalose 20 30 180 3 H1 #2 trehalose 20 50129 4 H2 #2 trehalose 20 50 53 4 J1 #2 trehalose 40 30 347 2 J2 #2trehalose 40 30 546 2 K1 #2 trehalose 40 50 377 4 K2 #2 trehalose 40 50198 4 L1 #2 trehalose 60 30 1451 1 L2 #2 trehalose 60 30 1164 1 M1 #2trehalose 60 50 409 3 M2 #2 trehalose 60 50 336 3

EXAMPLE 2

The following studies were carried out to assess the ability to delivera powdered nucleic acid composition to test subjects in vivo using themethods of the invention.

Plasmid Vector Construct: The pGREEN-1 vector construct, which containsthe Green Fluorescent Protein (GFP) gene under the control of a CMVpromoter, was used so that gene expression could be assessed directly byUV microscopy of histological sections from treated tissue samples.

Powdered Nucleic Acid Compositions: A powdered nucleic acid compositionwas prepared as follows. A mixture was formed by combining pGREEN-1vector plasmid with trehalose sugar to obtain a 1 μg:1 mg (w/w)DNA-sugar composition. This composition was lyophilized, compressed,ground, and then sieved, using the techniques described hereinabove. Theresulting condensed nucleic acid composition had an average particlesize ranging from about 38-75 μm.

Administrations: C57BL/10 mice were treated with 1 mg of the particulatecomposition via needleless injection. The composition was delivered to asuitably prepared target skin surface, and histological sections weretaken from the target site 24 hours after administration. GFP expressionwas determined directly using UV microscopy. As a result of theadministrations, GFP expression was seen in the treated skin tissue,confirming successful in vivo delivery of the powdered nucleic acidcomposition to the target skin, and the subsequent transfection of hostcells and expression of the GFP gene therefrom.

In another study, plasmids containing either a human Growth Hormone(hGH) or β-galactosidase (β-Gal) expression cassette were lyophilizedwith trehalose excipient to form nucleic acid formulations, which werecompressed, ground, and then sieved, using the above-describedtechniques. The resulting condensed nucleic acid compositions had anaverage particle size ranging from about 38-75 μm.

Female pigs (weighing 20-25 kg) were anesthestised with halothane, andthe belly skin was clipped to reveal a suitable target site. The abovepowdered nucleic acid compositions were individually administered to theprepared target site in 0.1 μg (hGH) or 1 μg (β-Gal) doses via aneedleless injection device (delivery pressure of 60 bar). The targetsites were biopsied 24 hours after treatment, and histological sectionswere analyzed for human growth hormone or β-Gal expression. Although nohGH expression was seen within the detection limits of the assay, amoderate degree of β-Gal expression was seen in the treated sites. Thelack of detectable hGH expression in this study is due, presumably, tothe low loading density of the nucleic acid (0.1 μg) in the composition.

EXAMPLE 3 Densification of Recombinant Human Growth Hormone (rhGH)

Lyophilized recombinant human growth hormone powder (Genotropin®,available from Pharmacia, Piscataway, N.J.) was obtained and reprocessedusing the method of the invention. Particularly, approximately 30 mg ofGenotropin was compacted under pressure using a Carver Laboratory PelletPress (Model 3620, available from Carver, Inc., Wabash, Ind.). Thepressure of compaction was 15,000 lbs/in², which was applied forapproximately 45 seconds. A pellet was obtained which was ground usingmortar and pestle until visually broken up. The resulting reduced pelletwas then sieved using a 53 μm sieve (Endecott, London). Particles havinga size greater than 53 μm were selected and appropriate dosages thereofwere measured into drug cassettes for delivery from a needlelesssyringe.

EXAMPLE 4 Visual Assessment of rhGH Particle Penetration

Lyophilized recombinant human growth hormone (rhGH) powder, anddensified rhGH particles prepared as described in Example 3 wereadministered to porcine subjects using a needleless syringe. The degreeof particle penetration was visually ascertained as follows.

Genotropin® 36 IU lyophilized powder was milled gently, weighed intoindividual doses of approximately 0.8 mg powder and loaded into aneedleless syringe device for delivery. Densified Genotropin® wasprepared as described in Example 3 and approximately 0.8 mg of thedensified particles were loaded into a needleless syringe device fordelivery.

Porcine subjects were prepared by clipping a sufficient area on thehindquarters. The lyophilized powder and densified particles weredelivered to the—porcine skin under high velocity. Upon a side-by-sidecomparison, it was observed that a higher proportion of the densifiedparticles penetrated the skin as evidenced by the visual presence of thelyophilized powder remaining largely on the surface of the skin whilesubstantially no densified particles were observed to remain on thesurface of the skin.

EXAMPLE 5 Serum Levels of Transdermally-Delivered rhGH

In order to determine the efficiency with which densified rhGH isdelivered using a needleless syringe system, the following study wascarried out. Three groups of 5 healthy New Zealand White rabbits wereprepared by clipping the fur from the flank area to expose a sufficientarea for delivery of lyophilized recombinant human growth hormone powder(rhGH) or densified rhGH by needleless syringe.

Approximately 0.8 mg of lyophilized Genotropin® powder was reconstitutedinto 1.8 mL of a suitable buffer without preservatives (e.g., sterilephosphate buffered saline (PBS)) to provide a Genotropin® solutionhaving a concentration of 20 IU/mL. 1 mL dosages were withdrawn bysyringe aim gently mixed with 1 mL of buffer to provide an injectionsolution having a concentration of 10 IU/mL.

Each animal in the first group were given 0.1 mL/kg of the injectionsolution by subcutaneous needle and syringe injection, and the injectionsite was observed to ensure that there was no leakage of the injectedsolution after administration. Venous blood samples were taken from themarginal ear vein of the right ear of each animal at 0, 30 minutes, 1,2, 4, 6, 12 and 24 hours after administration. Serum levels of rhGH wereascertained using an immunoassay with labeled anti-rhGH antibodies. Themean serum levels of subcutaneously delivered Genotropi® (●) found inthe animals of Group 1 at each time point are depicted in FIG. 4.

Approximately 2 mg of lyophilized Genotropin® powder was loaded into aneedleless syringe. The lyophilized powder was administered to eachanimal in the second group by needleless injection at high velocity.Venous blood samples were taken from the marginal ear vein of the rightear of each animal at 0, 30 minutes, 1, 2, 4, 6, 12 and 24 hours afteradministration. Serum levels of the administered rhGH were ascertainedusing an immunoassay with labeled anti-hGH antibodies. The mean serumlevels of transdermally injected lyophilized Genotropin® powder (▴)found in the animals of Group 2 at each time point are depicted in FIG.4.

Approximately 2 mg of densified Genotropin® particles prepared as inExample 3 was loaded into a needleless syringe. The densified particleswere administered to each animal in the third group by needlelessinjection at high velocity. Venous blood samples were taken from themarginal ear vein of the right ear of each animal at 0, 30 minutes, 1,2, 4, 6, 12 and 24 hours after administration. Serum levels of theadministered rhGH were ascertained using an immunoassay with labeledanti-rhGH antibodies. The mean serum levels of transdermally injecteddensified Genotropin® particles (▪) found in the animals of Group 3 ateach time point are depicted in FIG. 4.

As can be seen, markedly increased blood serum levels of the densifiedGenotropin® particles administered by needleless syringe were obtainedas compared to the lyophilized Genotropin® powder.

EXAMPLE 6 Determination of Optimum Conditions for Needleless SyringeDelivery of rhGH

In order to determine optimum conditions for delivery of rhGH using aneedleless syringe delivery system, the following study is carried out.One group of 8 healthy New Zealand White rabbits (2±0.25 kg) areprepared by clipping the fur from the flank area to expose a sufficientarea for delivery of Lyophilized recombinant human growth hormone powder(rhGH) or densified rhGH by needleless syringe, sub-cutaneous (SC) orintravenous (IV) injection. The animals are weighed at the start of thestudy and on a weekly basis throughout the study to determineappropriate Genotropin® dosages. The animals remain in one large groupto obtain statistically-significant data.

For an initial needleless syringe injection series, Genotropin® 36 IUlyophilized powder is milled gently and filled into a glass vial. Themilled lyophilized powder is weighed into individual dosages and loadedinto needleless syringe devices at approximately 0.8 mg powder/kg. Theinjection is conducted in the first week of the study, and multiplevenous blood samples (1 mL whole blood) are taken from the marginal earvein at times 0, 30 minutes, 1, 2, 4, 6, 12, 24 and 48 hours afteradministration. The animals are individually-housed at all times withfood and water available ad libitum.

Blood samples are handled and processed as follows: each venous bloodsample is allowed to clot at ambient temperature for approximately 30minutes and then left for an additional 30 minutes at approximately 4°C. Clotted samples are centrifuged for 10 minutes and the serum isaspirated and stored at −20° C. for analysis.

In the second week of the study, (approximately 1 week following theinitial needleless injection), the animals are administered aGenotropin® formulation prepared as follows: 36 IU (approximately 30 mg)of the lyophilized Genotropin® powder is reconstituted into 1.8 mL of asuitable buffer without preservatives (e.g., sterile phosphate bufferedsaline (PBS)) to provide a Genotropin® solution having a concentrationof 20 IU/mL. 1 mL dosages are withdrawn by syringe and gently mixed with1 mL of buffer to provide an injection solution having a concentrationof 10 IU/mL. Each animal is given an IV injection of 0.1 mL/kg of thesolution in the left ear.

Following IV injection, multiple venous blood samples are taken from themarginal ear vein of the right ear at times 0, 5, 10, 30 minutes, 1, 2,4, 6 and 12 hours after injection.

In the third week of the study, the animals are administered 0.1 mL/kgof a buffered Genotropin® solution (prepared as above) by sub-cutaneousinjection. The injection site is observed to ensure that there is noleakage after administration. Following the SC injections, multiplevenous blood samples are taken from the marginal ear vein of the rightear at times 0, 30 minutes, 1, 2, 4, 6, 12, 24 and 48 hours afteradministration.

In the fourth week of the study, approximately 0.8 mg powder/kg ofdensified Genotropin® particles (prepared as described in Example 3) areadministered to each animal using a needleless syringe, and multiplevenous blood samples are taken from the marginal ear vein of the rightear at times 0, 30 minutes, 1, 2, 4, 6, 12, 24 and 48 hours afteradministration.

Serum rhGH levels are determined as previously described andpharmacokinetic variables are calculated for each drug administrationtechnique. It is expected that needleless syringe administration of thedensified Genotropin® particles will result in achieving and maintainingin vivo therapeutic levels of the administered drug.

EXAMPLE 7 Bio-Activity of Densified rhGH Delivered in vivo to GrowthHormone-Deficient Rats

To evaluate the bio-activity of recombinant human growth hormone thathas been densified using the method of the present invention thefollowing study is carried out.

Dwarf or hypophysectomized (growth hormone-deficient) rats areadministered pharmaceutical preparations containing either: densifiedGenotropin® particles; lyophilized Genotropin® powder; or placebo atapproximately 4 IU rhGH per animal/week by daily subcutaneous (SC)injection. In particular, on 5 successive days, fur from the peritonealregion of the animal subjects is clipped prior to administration of thedensified rhGH, the lyophilized rhGH or the placebo by SC injection.Body weight and, if desired, bone size and length are monitored on adaily basis.

Bio-activity of the densified rhGH particle formulation is determined bymeasuring body weight change over time. It is expected that thedensified rhGH particle formulation will retain sufficient bio-activity.

EXAMPLE 8 Densification of Commonly Used Excipients

Finely ground powders of pharmaceutical grade mannitol and lactose wereobtained and reprocessed using the method of the invention.Particularly, approximately 30 to 50 mg of mannitol or lactose werecompacted under pressure using a Carver Laboratory Pellet Press (Model3620, available from Carver, Inc., Wabash, Ind.). The pressure ofcompaction was 10,000 lbs/in² which was applied for approximately 30seconds. The resulting compacted pellets were ground using mortar andpestle until visually broken up, and then sieved to select for particleshaving a size greater than about 50 μm using the methods described inExample 3. In both the mannitol and the lactose preparations, asignificant size reduction was observed when the compacted particleswere compared against like weights of the non-densified startingmaterials.

EXAMPLE 9 Quantification of Densified Excipients

Pharmaceutical grade trehalose and mannitol excipients were obtained andprocessed according to the method of the invention. Both excipientpreparations were processed in several different ways, and absolutedensity, envelope density, and average particle size of the resultantpreparations were measured as described below.

Trehalose 45H3830 (Sigma) and mannitol K91698380-703 (Merck) were eithersieved, or freeze dried, compacted, ground and then sieved. A range ofthe resulting preparations were then analyzed for particle size anddensity measurements.

Portions of each sugar excipient were sieved to obtain preparationshaving reduced particle sizes. Sieving was carried out using stainlesssteel sieves for two hours at 3 mm amplitude using three sieve sizes (75μm, 53 μm and 38 μm) without additional processing.

Alternatively, portions of each sugar excipient were processed using themethods of the invention as follows. 40 g of each of the sugars wasdissolved in water, flash frozen, and then freeze dried over night.Samples of each freeze dried preparation were retained, and theremainder compacted in a 13 mm compression die (15,000 lbs/inch² for 45seconds) into discs. The mannitol discs were ground using mortar andpestle, and then sieved as described above at 3 mm amplitude, usingthree sieve sizes. The trehalose discs were first ground in a vibratoryball mill (Retsch mill), then ground by mortar and pestle and sieved asabove.

Samples from each of the above-described excipient preparations werethen analyzed for absolute and envelope density. Absolute density wasdetermined using helium pycnometry, and envelope density was determinedusing a GeoPyc™ Model 1360 Envelope Density Analyzer (MicromeriticsInstrument Corp.). The results of the analysis are depicted below inTable 4. TABLE 4 Trehalose Density Mannitol Density (g/cm³) (g/cm³)Pretreatment Absolute Envelope Absolute Envelope Sieved 1.5 0.5 1.5 0.5Freeze Dried 1.5 0.3 1.5 0.3 Freeze 1.5 0.8 1.5 0.8 Dried, Compressed,Milled, Sieved

As can be seen in Table 4, none of the various processing methods had asignificant effect on the absolute density of the powdered excipients.Further, as expected, the non-compacted, freeze-dried sugars had a muchlower envelope density than the other preparations, and a concomitantlyhigher porosity (measurement not shown). The density measurements forthe trehalose and mannitol samples clearly demonstrate that the methodsof the invention (compression, milling, sieving) lead to a significantincrease in envelope density relative to both the freeze-dried and thesieved preparations. These results indicate that the novel methodsdescribed herein can be used to provide densified particulatepharmaceutical preparations that are suitable for delivery vianeedleless injection techniques.

Accordingly, novel methods for DNA delivery as well novel methods fordensifying particulate pharmaceutical compositions, and densifiedpharmaceutical compositions formed therefrom, have been described.Although preferred embodiments of the subject invention have beendescribed in some detail, it is understood that obvious variations canbe made without departing from the spirit and the scope of the inventionas defined by the appended claims.

1. A method for delivering densified particles to a target tissue orcell, the method comprising the steps of: (i) forming densifiedparticles from a particulate pharmaceutical preparation, the method forforming the densified particles comprising compacting the preparation toprovide a compacted pharmaceutical preparation and size-reducing thecompacted preparation into densified particles of suitable size anddensity for transdermal delivery thereof by needleless injection; and(ii) administering said densified particles to the target tissue or cellby needleless injection.
 2. The method of claim 1, wherein the particleshave an average size predominantly in the range of about 10 to 250 μm.3. The method of claim 1, wherein the particles are delivered to a cellin epidermal tissue.
 4. The method of claim 1, wherein the particles aredelivered to a cell in the stratum basal layer of skin tissue.
 5. Themethod of claim 1, wherein the particles are comprised of a nucleic acidmolecule and a pharmaceutically acceptable excipient.
 6. The method ofclam 1, wherein the particles are delivered to the target tissue or cellin vivo or ex vivo.
 7. The method of claim 1, wherein the nucleic acidmolecule comprises a nucleotide sequence encoding an immunogen.
 8. Amethod for forming densified particles from a particulate pharmaceuticalpreparation, comprising compacting the preparation to provide acompacted pharmaceutical preparation and size-reducing the compactedpreparation into densified particles of suitable size and density fortransdermal delivery thereof by needleless injection.
 9. A methodaccording to claim 8, wherein the suitable size is in the range of about0.1 to 150 μm mean diameter.
 10. A method according to claim 8, whereinthe densified particles have a particle density in the range of about0.5 to 3.0 g/cm³.
 11. A method according to claim 8, wherein sizereducing of the compacted material is carried out by milling and/orsieving.
 12. A method according to claim 8, wherein the method furthercomprises selecting densified particles using size classification.
 13. Amethod according to claim 8, wherein the size classification of thedensified particles is carried out using sieving or cyclone separation.14. A method according to claim 8, wherein the particulatepharmaceutical preparation is a preparation of a gene construct.
 15. Adensified particulate pharmaceutical composition formed from alyophilised or spray-dried pharmaceutical preparation, said densifiedcomposition having an average particle size in the range of about 0.1 to250 μm mean diameter and a particle density in the range of 0.1 to 25g/cm³.
 16. A composition according to claim 15, wherein the lyophilisedor spray-dried pharmaceutical preparation is a heat-sensitivebiopharmaceutical preparation.
 17. A composition according to claim 15,wherein the lyophilised or spray-dried pharmaceutical preparation is apreparation of a peptide or protein.
 18. A composition according toclaim 15, wherein the particulate pharmaceutical preparation is apreparation of a gene construct.
 19. A composition according to claim15, wherein the particle size is in the range of about 0.1 to 150 μmmean diameter.
 20. A composition according to claim 15, wherein theparticle density is in the range of about 0.5 to 3.0 g/cm³.